Lipid-Based Nanocarriers for Delivery of Neuroprotective Kynurenic Acid: Preparation, Characterization, and BBB Transport.

Encapsulation possibilities of an extensively investigated neuroprotective drug (kynurenic acid, KYNA) are studied via lipid-based nanocarriers to increase the blood-brain barrier (BBB) specific permeability. The outcomes of various preparation conditions such as stirring and sonication time, concentration of the lipid carriers and the drug, and the drug-to-lipid ratio are examined. Considering the experimentally determined encapsulation efficiency, hydrodynamic diameter, and ζ-potential values, the initial lipid and drug concentration as well as the stirring and sonication time of the preparation were optimized. The average hydrodynamic diameter of the prepared asolectin-(LIP) and water-soluble lipopolymer (WSLP)-based liposomes was found to be ca. 25 and 60 nm under physiological conditions. The physicochemical characterization of the colloidal carriers proves that the preparation of the drug-loaded liposomes was a successful process, and secondary interactions were indicated between the drug molecule and the polymer residues around the WSLP membrane. Dissolution profiles of the active molecule under physiological conditions were registered, and the release of the unformulated and encapsulated drug is very similar. In addition to this outcome, the in vitro polar brain lipid extract (porcine)-based permeability test proved the achievement of two- or fourfold higher BBB specific penetration and lipid membrane retention for KYNA in the liposomal carriers relative to the unformatted drug.

The Effect of Concentration, Temperature, and pH on the Formation of Hyaluronic Acid-Surfactant Nanohydrogels.

The assembly of colloidal hyaluronic acid (HyA, as a polysaccharide) based hydrogel particles in an aqueous medium is characterized in the present paper, with an emphasis on the particular case of nanohydrogels formed by surfactant-neutralized polysaccharide networks. The structural changes and particle formation process of polysaccharide- and cationic-surfactant-containing systems were induced by the charge neutralization ability and the hydrophobic interactions of cetyltrimethylammonium bromide (CTAB) under different conditions. Based on the rheological, light scattering, ζ-potential, turbidity, and charge titration measurements, it can be concluded that the preparation of the HyA-CTAB particles can be greatly controlled. The results indicate that more available negative charges can be detected on the polymer chain at smaller initial amounts of HyA (cHyA < 0.10 mg/mL), where a molecular solution can be formed. The change in the pH has a negligible effect on the formation process (particle aggregation appears at nCTAB/nHyA,monomer~1.0 in every case), while the temperature dependence of the critical micelle concentration (c.m.c.) of CTAB determines the complete neutralization of the forming nanohydrogels. The results of our measurements confirm that after the appearance of stable colloidal particles, a structural change and aggregation of the polymer particles take place, and finally the complete charge neutralization of the system occurs.

The Mycoplasma hyorhinis genome displays differential chromatin accessibility.

Whilst the regulation of chromatin accessibility and its effect on gene expression have been well studied in eukaryotic species, the role of chromatin dynamics and 3D organisation in genome reduced bacteria remains poorly understood [1,2]. In this study we profiled the accessibility of the Mycoplasma hyorhinis genome, these data were collected fortuitously as part of an experiment where ATAC-Seq was conducted on mycoplasma, contaminated mammalian cells. We found a differential and highly reproducible chromatin accessibility landscape, with regions of increased accessibility corresponding to genes important for the bacteria's life cycle and infectivity. Furthermore, accessibility in general correlated with transcriptionally active genes as profiled by RNA-Seq, but peaks of high accessibility were also seen in non-coding and intergenic regions, which could contribute to the topological organisation of the genome. However, changes in transcription induced by starvation or application of the RNA polymerase inhibitor rifampicin did not themselves change the accessibility profile, which confirms that the differential accessibility is inherently a property of the genome, and not a consequence of its function. These results together show that differential chromatin accessibility is a key feature of the regulation of gene expression in bacteria.

Core-Shell Structured PLGA Particles Having Highly Controllable Ketoprofen Drug Release.

The non-steroid anti-inflammatory drug ketoprofen (KP) as a model molecule is encapsulated in different poly(lactide-co-glycolide) (PLGA) nanostructured particles, using Tween20 (TWEEN) and Pluronic F127 (PLUR) as stabilizers to demonstrate the design of a biocompatible colloidal carrier particles with highly controllable drug release feature. Based on TEM images the formation of well-defined core-shell structure is highly favorable using nanoprecipitation method. Stabile polymer-based colloids with ~200-210 nm hydrodynamic diameter can be formed by successful optimization of the KP concentration with the right choice of stabilizer. Encapsulation efficiency (EE%) of 14-18% can be achieved. We clearly confirmed that the molecular weight of the stabilizer thus its structure greatly controls the drug release from the PLGA carrier particles. It can be determined that ~20% and ~70% retention is available with the use of PLUR and TWEEN, respectively. This measurable difference can be explained by the fact that the non-ionic PLUR polymer provides a steric stabilization of the carrier particles in the form of a loose shell, while the adsorption of the non-ionic biocompatible TWEEN surfactant results in a more compact and well-ordered shell around the PLGA particles. In addition, the release property can be further tuned by decreasing the hydrophilicity of PLGA by changing the monomer ratio in the range of ~20-60% (PLUR) and 70-90% (TWEEN).

Evaluation of noble metal nanostructure-serum albumin interactions in 2D and 3D systems: Thermodynamics and possible mechanisms.

In this review, we clearly highlight the importance of the detailed study of the interactions between noble metal colloids (nanoparticles (NPs) and nanoclusters (NCs)) with serum albumins (SAs) due to their rapidly growing presence in biomedical research. Besides the changes in the structure and optical property of SA, we demonstrate that the characteristic localized surface plasmon resonance (LSPR) feature of the colloidal noble metal NPs and the size- and structure-dependent photoluminescence (PL) property of the sub-nanometer sized NCs are also altered differently because of the interactions between them. Namely, for plasmonic NPs - SA interactions the PL quenching of SA (mainly static) is identified, while the SA cause PL enhancement of the ultra-small NCs after complexation. This review summarizes that the thermodynamic nature and the possible mechanisms of the binding processes are dependent partly on the size, morphology, and type of the noble metals, while the chemical structure as well as the charge of the stabilizing ligands have the most dominant effect on the change in optical features. In addition to the thermodynamic data and proposed binding mechanisms provided by three-dimensional spectroscopic techniques, the quantitative and real-time data of "quasi" two-dimensional sensor apparatus should also be considered to provide a comprehensive evaluation on many aspects of the particle/cluster - SA interactions.

Prodrugs of E-selectin Antagonists with Enhanced Pharmacokinetic Properties.

Because of their large polar surface area, carbohydrates often exhibit insufficient pharmacokinetic properties. Specifically, the carboxylic acid function of the tetrasaccharide sialyl Lewisx , a pharmacophore crucial for the formation of a salt bridge with selectins, prevents oral availability. A common approach is the transfer of carboxylic acid into ester prodrugs. Once the prodrug is either actively or passively absorbed, the active principle is released by hydrolysis. In the present study, ester prodrugs of selectin antagonists with aliphatic promoieties were synthesized and their potential for oral availability was investigated in vitro and in vivo. The addition of lipophilic ester moieties to overcome insufficient lipophilicity improved passive permeation into enterocytes, however at the same time supported efflux back to the small intestines as well as oxidation into non-hydrolysable metabolites. In summary, our examples demonstrate that different modifications of carbohydrates can result in opposing effects and have to be studied in their entirety.

Detailed Calorimetric Analysis of Mixed Micelle Formation from Aqueous Binary Surfactants for Design of Nanoscale Drug Carriers.

While numerous papers have been published according to the binary surfactant mixtures, only a few articles provide deeper information on the composition dependence of the micellization, and even less work attempts to apply the enhanced feature of the mixed micelles. The most important parameter of the self-assembled surfactants is the critical micelle concentration (cmc), which quantifies the tendency to associate, and provides the Gibbs energy of micellization. Several techniques are known for determining the cmc, but the isothermal titration calorimetry (ITC) can be used to measure both cmc and enthalpy change (ΔmicH) accompanying micelle formation. Outcomes of our calorimetric investigations were evaluated using a self-developed routine for handling ITC data and the thermodynamic parameters of mixed micelle formation were obtained from the nonlinear modelling of temperature- and composition- dependent enthalpograms. In the investigated temperature and micelle mole fractions interval, we observed some intervals where the cmc is lower than the ideal mixing model predicted value. These equimolar binary surfactant mixtures showed higher solubilization ability for poorly water-soluble model drugs than their individual compounds. Thus, the rapid and fairly accurate calorimetric analysis of mixed micelles can lead to the successful design of a nanoscale drug carrier.

The pH-Dependent Controlled Release of Encapsulated Vitamin B1 from Liposomal Nanocarrier.

In this work, we firstly presented a simple encapsulation method to prepare thiamine hydrochloride (vitamin B1)-loaded asolectin-based liposomes with average hydrodynamic diameter of ca. 225 and 245 nm under physiological and acidic conditions, respectively. In addition to the optimization of the sonication and magnetic stirring times used for size regulation, the effect of the concentrations of both asolectin carrier and initial vitamin B1 on the entrapment efficiency (EE %) was also investigated. Thermoanalytical measurements clearly demonstrated that after the successful encapsulation, only weak interactions were discovered between the carriers and the drug molecules. Moreover, the dissolution profiles under physiological (pH = 7.40) and gastric conditions (pH = 1.50) were also registered and the release profiles of our liposomal B1 system were compared with the dissolution profile of the pure drug solution and a manufactured tablet containing thiamin hydrochloride as active ingredient. The release curves were evaluated by nonlinear fitting of six different kinetic models. The best goodness of fit, where the correlation coefficients in the case of all three systems were larger than 0.98, was reached by application of the well-known second-order kinetic model. Based on the evaluation, it was estimated that our liposomal nanocarrier system shows 4.5-fold and 1.5-fold larger drug retention compared to the unpackaged vitamin B1 under physiological conditions and in artificial gastric juice, respectively.

Adenosine integrates light and sleep signalling for the regulation of circadian timing in mice.

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A1/A2A receptor signalling through the activation of the Ca2+ -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.

Serum protein-hyaluronic acid complex nanocarriers: Structural characterisation and encapsulation possibilities.

A protein-polysaccharide-based potential nanocarrier system have been developed via a simple, one-step preparation protocol without the use of long-term heating and the utilization of hardly removable crosslinking agents, surfactants, and toxic organic solvents. To the best of our knowledge, this article is the first which summarizes in detail the pH-dependent quantitative relationship between the bovine serum albumin (BSA) and hyaluronic acid (HyA) confirmed by several physico-chemical techniques. The formation of colloidal complex nanoconjugates with average diameter of ca. 210-240 nm is strongly depend on the pH and the applied BSA:HyA mass ratio. Particle charge titrations studies strongly support the core-shell type structure, where the BSA core is covered by a thick HyA shell. Besides the optimization of these conditions, the drug encapsulation capacity and the dissolution profiles have been also studied for ibuprofen (IBU) and 2-picolinic acid (2-PA) as model drugs.

Increased miR-20b Level in High Grade Cervical Intraepithelial Neoplasia.

Cervical cancer is a common malignant tumor worldwide ranking fourth in incidence and mortality among females, which was reduced significantly by cytology screening and human papilloma virus (HPV) DNA testing. The specificity of cytology is high; however, the sensitivity is low, in contrast to the HPV DNA testing. Despite the success of these measures, new biomarkers are still considered to aim increasing sensitivity and specificity of screening and diagnosis. Significant alterations in microRNA (miRNA) expression have been detected in several cancers with variable consistency. To investigate the stratification role of miRNAs between normal epithelium and cervical intraepithelial neoplasia (CIN2-3), we screened the expression of 667 miRNAs to identify significant markers (n = 10), out of them 9 miRNAs were applied in the study (miR-20b, -24, -26a, -29b, -99a, -100, -147, -212, -515-3p) along with RNU48 and U6 as the references. To benchmark the miRNAs, 22 paired (tumor-free and tumor tissue pairs) laser microdissection-obtained cervical formalin fixed, paraffin embedded tissue samples were assayed. The expression of miR-20b was 2.4 times higher in CIN2-3 samples as compared to normal tissues (p < 0.0001). In the HPV16-positive subsets of the samples (n = 13), miR-20b showed 2.9-times elevation (p < 0.001), whereas miR-515 was 1.15-times downregulated (p < 0.05) in CIN2-3 as compared to normal tissue. These results suggest the potential value of miR-20b as a statification biomarker in order to differentiate neoplastic and non-tumorous cases.

Chitosan-modified hyaluronic acid-based nanosized drug carriers.

Fabrication possibilities, detailed size and structural characterization of biodegradable chitosan (Chit) polysaccharide-modified hyaluronic acid (HyA)-based colloidal carriers are demonstrated. The negatively charged and highly hydrophilic HyA polymer chains have been ionically modified by positively charged pure Chit and crosslinked Chit macromolecules at various Chit/HyA weight ratios, which resulted in the formation of carrier nanoparticles (NPs) having three different nanostructures depending on the polymer concentrations. Electrostatically-compensated Chit/HyA polymer coils with loose colloidal structure, tripolyphosphate (TPP)-crosslinked Chit-TPP/HyA NPs having interpenetrating polymer network and well-defined Chit-TPPcore-HyAshell NPs with diameters of 100-300 nm were also prepared and were loaded with tocopherol (TCP) and cholecalciferol (D3) having Vitamin E and D activity, respectively. By using rheological, particle charge titration and conductivity studies we first confirmed that the expected 1:1 Chit/HyA monomer molar ratio is strongly influenced by the pH of the polymer solutions as well as the deacetylation degree of Chit which are crucial factors for the solubility, purity and the quality of the commercially available biocompatible Chit in aqueous medium. Encapsulation studies revealed that D3 could be better incorporated in every system, especially in Chit-TPP/HyA NPs, while for TCP the simple Chit/HyA polymer coils were the most promising carriers.

Vitamin E-Loaded PLA- and PLGA-Based Core-Shell Nanoparticles: Synthesis, Structure Optimization and Controlled Drug Release.

The (±)-α-Tocopherol (TP) with vitamin E activity has been encapsulated into biocompatible poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) carriers, which results in the formation of well-defined nanosized (d ~200-220 nm) core-shell structured particles (NPs) with 15-19% of drug loading (DL%). The optimal ratios of the polymer carriers, the TP active drug as well as the applied Pluronic F127 (PLUR) non-ionic stabilizing surfactant, have been determined to obtain NPs with a TP core and a polymer shell with high encapsulation efficiency (EE%) (69%). The size and the structure of the prepared core-shell NPs as well as the interaction of the carriers and the PLUR with the TP molecules have been determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), infrared spectroscopy (FT-IR) and turbidity studies, respectively. Moreover, the dissolution of the TP from the polymer NPs has been investigated by spectrophotometric measurements. It was clearly confirmed that increase in the EE% from ca. 70% (PLA/TP) to ca. 88% (PLGA65/TP) results in the controlled release of the hydrophobic TP molecules (7 h, PLA/TP: 34%; PLGA75/TP: 25%; PLGA65/TP: 18%). By replacing the PLA carrier to PLGA, ca. 15% more active substance can be encapsulated in the core (PLA/TP: 65%; PLGA65/TP: 80%).

Modelling the neuropathology of lysosomal storage disorders through disease-specific human induced pluripotent stem cells.

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches.

The effect of synthesis conditions and tunable hydrophilicity on the drug encapsulation capability of PLA and PLGA nanoparticles.

Three drugs with different hydrophilicity are encapsulated in poly-lactide (PLA) and Poly(lactide-co-glycolide) (PLGA) drug delivery systems prepared by ring-opening polymerization (ROP). Formation of well-defined core-shell type nanoparticles (NPs) is observed for α-tocopherol (TP) and by systematically altering the hydrophilicity of the drug carrier NPs the entrapment efficiency (EE (%)) can be remarkably controlled. The highest (90%) of EE (%) is obtained for the most lipophilic TP from the applied three drugs in the 75% lactide-containing PLGA75 NPs, which is ca. 69% for PLA NPs. Subsequent to drug loading the detailed characterization of the polymers and the formed NPs was carried out. Precipitation titrations reveal that our PLGAs have narrower weight distribution than the commercially available polymer enabling favorable properties to obtain NPs with better size distribution. It is pointed out that during the synthesis the applied solvent and stabilizing agent play a decisive role in the size distribution and stability of the drug carrier NPs. The Pluronic F127-stabilized NPs have the smallest diameter (ca. 190 nm) with less polydispersity among the applied stabilizing agent in nanoprecipitation.

On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin.

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.

Cross-linked and hydrophobized hyaluronic acid-based controlled drug release systems.

This work demonstrates the preparation, structural characterization, and the kinetics of the drug release of hyaluronic acid (HyA)-based colloidal drug delivery systems which contain hydrophobic ketoprofen (KP) as model molecule. Because of the highly hydrophilic character of HyA the cross-linked derivatives at different cross-linking ratio have been synthesized. The hydrophobized variants of HyA have also been produced by modifying the polymer chains with cetyltrimethylammonium bromide (CTAB) at various HyA/CTAB ratios. Due to modifications the coherent structure of HyA changes into an incoherent colloidal system that were verified by rheological investigations. Nearly 70% of the encapsulated KP dissolve from the totally cross-linked HyA carrier but the release rate of KP is about 20% (after 8 h) from the CTAB-modified colloidal system at HyA monomer/CTAB 1:0.8 mass ratio. It has been verified that the modified HyA may be a potential candidate for controlled drug release of hydrophobic KP molecules.

Rational-Differential Design of Highly Specific Glycomimetic Ligands: Targeting DC-SIGN and Excluding Langerin Recognition.

At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carbohydrate-based PAMPS or DAMPS (pathogen- or danger-associated molecular patterns, respectively) and promote immune response regulation. However, some CLRs are hijacked by viral and bacterial pathogens. Thus, the design of ligands able to target specifically one CLR, to either modulate an immune response or to inhibit a given infection mechanism, has great potential value in therapeutic design. A case study is the selective blocking of DC-SIGN, involved notably in HIV trans-infection of T lymphocytes, without interfering with langerin-mediated HIV clearance. This is a challenging task due to their overlapping carbohydrate specificity. Toward the rational design of DC-SIGN selective ligands, we performed a comparative affinity study between DC-SIGN and langerin with natural ligands. We found that GlcNAc is recognized by both CLRs; however, selective sulfation are shown to increase the selectivity in favor of langerin. With the combination of site-directed mutagenesis and X-ray structural analysis of the langerin/GlcNS6S complex, we highlighted that 6-sulfation of the carbohydrate ligand induced langerin specificity. Additionally, the K313 residue from langerin was identified as a critical feature of its binding site. Using a rational and a differential approach in the study of CLR binding sites, we designed, synthesized, and characterized a new glycomimetic, which is highly specific for DC-SIGN vs langerin. STD NMR, SPR, and ITC characterizations show that compound 7 conserved the overall binding mode of the natural disaccharide while possessing an improved affinity and a strict specificity for DC-SIGN.

Formation of mono- and binuclear neodymium(iii)-gluconate complexes in aqueous solutions in the pH range of 2-8.

The complex formation between Nd(iii) and d-gluconate (Gluc-) is of relevance in modelling the chemical equilibria of radioactive waste repositories. In the present work, the formation of NdpGlucqH-r complexes at 25 °C and pH = 2-8 was studied via spectrophotometry, potentiometry, freezing point depression, conductometry and NMR spectroscopy. In addition to the four mononuclear complexes (pq-r = 110, 120, 130 and 11-2), the formation of two binuclear, so far unknown complexes (pq-r = 23-2 and 24-2) was revealed. Between pH = 5.5 and 7, with the increasing metal ion and ligand concentrations, the Nd2Gluc3H-2+ species becomes progressively predominant. Under the conditions characteristic of waste repositories, however, the formation of these complexes can be neglected. Regarding the binding sites of Gluc-, C2-OH and C3-OH groups, in addition to the carboxylate ion, were identified from 1H and 13C spectroscopic measurements. Above pH = 6, the metal-ligand interactions became stronger implying the formation of deprotonated complexes involving the C2-OH group, while the displacement of the second proton at the C3-OH is also possible. The metal ion induced deprotonation of the ligand was confirmed by DFT calculations.